Southern Blot Data Research Articles

Southern analysis of BT-R1, the Manduca sexta gene encoding the receptor for the Cry1Ab toxin of Bacillus thuringiensis.

Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210 kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp, berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210 kDa protein, termed BT-R1 (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R1, the gene encoding the 210 kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R1 as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R1. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R1, whereas the other is a closely related homologue.

FISH identifies different types of duplications with 12q13-15 as the commonly involved segment in B-cell lymphoproliferative malignancies characterized by partial trisomy 12

Clinical, cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data of 18 patients with different subtypes of B-cell non-Hodgkin's lymphoma, cytogenetically characterized by partial trisomy 12, are presented. These chromosomal changes occurred predominantly in clinically progressive chronic lymphocytic leukemia, mixed cell type, and advanced-stage follicle center cell lymphoma at the time of relapse or transformation into diffuse large cell lymphoma. Partial trisomy 12 consistently included the long arm of chromosome 12, either completely or partially, and resulted from dup(12q) or other rearrangements involving chromosome 12. The duplications were cytogenetically identified as dup(12)(q13q23), dup(12)(q13q22), or dup(12)(q13q15) in follicle center cell lymphoma or t(14;18)-positive diffuse large cell lymphoma; dup(12)(q13q22) or dup(12)(q13q24) in chronic lymphocytic leukemia; and dup(12)(q13q21) in a case of t(14;18)-negative diffuse large cell lymphoma. FISH, using library probes and a panel of YAC probes, mapped along the long arm of chromosome 12, confirmed the cytogenetic results in all cases analyzed except for three cases of t(14;18)-positive follicle center lymphoma or diffuse large cell lymphoma with dup(12q). In these cases, FISH showed similar, possibly identical, duplications, which involved a region more centromeric (12q11-21) than assumed by karyotypic analysis (12q13-22 or 12q13-23) and included alphoid DNA sequences, a combination hitherto unknown. In addition, commonly duplicated regions of chromosome 12 could be defined: 12q11-21, including alphoid DNA sequences for follicle center cell lymphoma or t(14;18)-positive diffuse large cell lymphoma, 12q13-22 for chronic lymphocytic leukemia, and 12p13-q15 for marginal zone cell lymphoma, all of which overlapped in 12q13-15. Whether these regions, especially 12q13-15, may contain genes which are important in malignant transformation or disease progression of B-cell lymphoproliferative malignancies characterized by complete or partial trisomy 12 remains to be determined.

Structure and organization of the peridinin-chlorophyll a-binding protein gene in Gonyaulax polyedra.

We have identified a major 32-kDa protein in the dinoflagellate Gonyaulax polyedra as a peridinin-chlorophyll a-binding protein (PCP), based on micro-sequence data and immunological cross-reaction with antibodies raised against PCP from another dinoflagellate species. A cDNA for this protein, identified by a PCR-based cloning strategy, encoded all 68 of the amino acids microsequenced, thus confirming the identity of the clone. The PCP gene is highly expressed at both the mRNA and protein levels, and only PCP transcripts corresponding in size to the cDNA sequence were detected. Slot blot analyses show that there are roughly 5000 copies of the PCP gene in Gonyaulax, making this gene one of the most highly repeated protein-coding genes ever reported, yet the sequence of the different gene copies in the genome appears extraordinarily well conserved as judged by Southern blot analyses. The gene, as indicated by Southern blot and PCR data, is suggested to be present in 5000 intronless copies arranged head to tail in the genome, separated by conserved 1-kb spacers. Based on the conserved sequence of the spacer region, its presence next to each of the PCP coding sequences, and the uniform size of the PCP transcript, we propose that this region represents a dinoflagellate transcriptional promoter. This putative promoter region contains none of the sequence elements for DNA-binding proteins involved in transcriptional initiation reported in other organisms.

Characteristic pattern of chromosomal gains and losses in marginal zone B cell lymphoma detected by comparative genomic hybridization

Marginal zone B cell lymphoma (MZBCL) represents a distinct subtype of B cell non-Hodgkin's lymphoma, which has been recently recognized and defined as a disease entity. We investigated 25 cases (18 at primary diagnosis and seven during the course of disease) of MZBCL by comparative genomic hybridization (CGH) and compared these results with cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data. Twenty of the 25 cases (80%) showed gains (total 49) or losses (total 15) of genetic material. In extranodal, nodal, and splenic MZBCL, material of chromosomes 3 (52% of cases), 18 (32%), X (24%), and 1q (16%) was most frequently gained, whereas losses predominantly involved chromosomes 17 (16%) and 9 (12%). High-level amplifications involving the regions 18q21-23 and 18q21-22, respectively, were detected in two cases. Gains of chromosomes 1q and 8q and losses of chromosome 17 or 17p occurred more frequently in relapsed or progressive lymphomas. For all of the frequently affected chromosomes, CGH allowed narrowing of the relevant subregions including 3q21-23, 3q25-29 and 18q21-23. By Southern blot analysis, the BCL2, BCL6, and CMYC proto-oncogenes were found to be a part of the over-represented regions in two cases, one case, and two cases, respectively, with gains involving 18q, 3q or 8q. In 13 cases, CGH revealed chromosomal imbalances which were not detected by cytogenetic analysis but could be confirmed by FISH or Southern blot analysis in all cases investigated. On the other hand, CGH failed to detect trisomy 3, trisomy 18, and deletion 7q in three cases with a low proportion of tumor cells bearing these abnormalities, as shown by interphase FISH. The characteristic pattern of chromosomal gains and losses detected in this study confirms the distinct nature of MZBCL and may point to chromosomal regions involved in the pathogenesis of these neoplasms.

Expression, evolution and genomic complexity of potassium ion channel genes of Arabidopsis thaliana

Summary Potassium ion (K + ) channels are of major importance in several functionally diverse processes in plant physiology, including K + transport, membrane potential control, osmoregulation, and stomatal movement. In animals, K + channels are encoded by multigene families and are differentially expressed during development. Little is known about the genomic organisation, expression or evolution of plant K + channels. We report here, the partial sequence of a putative K+ channel cDNA from Arabidopsis (KAT2) showing sequence homology to previously identified voltage-gated K + channel genes KAT1 and AKT1, and which hybridises to a single 2.1 kb mRNA in leaves. The existence of KAT2, together with numerous similar sequences identified by Southern blot analyses, suggest that K + channels in Arabidopsis are encoded by multiple genes. Southern blot data also suggest that KAT and AKT homologs are present in many other plant species. Analysis of the expression pattern of KAT1, KAT2 and AKT1 demonstrate a tissue-specific regulation of K + channel genes in plants.

Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification

We have produced in transgenic maize seed the glycoprotein, avidin, which is native to avian, reptilian, and amphibian egg white. A transformant showing high-level expression of avidin was selected. Southern blot data revealed that four copies of the gene are present in this transformant. The foreign protein represents >2% of aqueous soluble extracted protein from populations of dry seed, a level higher than any heterologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, an organ representing only 12% of the dry weight of the seed. This indicates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference in the seed. The mature protein is primarily localized to the intercellular spaces. An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or total male sterility. Seed populations from transgenic plants were maintained by outcrossing and segregate 1:1 for the trait. In generations T2–T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extractable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or growing location. Cracked and flaked kernels stored at −29°C or 10 °C for up to three months showed no significant loss of avidin activity. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demonstrated that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation. This study shows that producing avidin from maize is not only possible but has practical advantages over current methods.

CDNA cloning of mouse nebulin. Evidence that the nebulin-coding sequence is highly conserved among vertebrates.

Nebulin is a family of giant myofibrillar proteins with molecular masses ranging over 700-900 kDa. Using a human nebulin cDNA probe, we isolated three nebulin cDNA clones from a mouse skeletal muscle cDNA library. These three clones, labeled 8c. 7a and 4b. carry inserts of 2.0, 3.0 and 3.5 kb, respectively. In Northern blots, each insert detected the same approximately = 25 kb message from skeletal muscle as the human nebulin probe, while detecting no messages from cardiac muscle. Sequence data in combination with reverse-transcriptase PCR indicates that clones 7a and 8c overlap to form 4076 bp contiguous sequence. Alignment with the published full-length human nebulin sequence indicates that clone 4b overlaps with clone 7a over 1596 bp. However, after the first 798-bp overlap, the sequence of these two mouse nebulin clones diverge, suggesting that they derive from distinct transcripts encoding isoforms of mouse nebulin. The mouse nebulin clones encode a series of = 245-residue super repeats, each of which can be subdivided into seven = 35-residue, weakly repeating modules centered around a conserved tyrosine residue, consistent with the human nebulin sequence. The mouse nebulin clones align along the central third of the full-length human sequence, corresponding to super repeats 8-16 of the 22 super repeats found in human nebulin. The translated sequence is greater than 90% identical to the human sequence, with the exception of a 200-amino-acid region at the C-terminus of clone 4b, which is less than 60% identical. In genomic Southern blots, a mouse nebulin probe detected a homologous sequence in a wide variety of vertebrate species under stringent conditions. However, no significant hybridization was observed to genomic DNA from invertebrates and microorganisms, even under very low stringency. The sequence and Southern-blot data suggest that the nebulin sequence is highly conserved among vertebrate species.

Restriction map of two yeast artificial chromosomes spanning the murine casein locus.

The caseins are the major mammalian milk proteins (reviewed by Mercier and Vilotte 1993), constituting a dietary supply of amino acids and calcium to the infant. In the presence of calcium, the so-called calcium-sensitive caseins of the a and [3 types form loose, non-crystalline aggregates termed micelles, which are stabilized by calcium-insensitive K casein. In the gut the site-specific cleavage of K casein by rennin causes the milk to clot and remain in the stomach, facilitating digestion. The caseins are encoded by a small gene family, which in cows and sheep consists of four members, ~X~l, [3, oLs2, and K, and in mice and rats five, c~, [3, 7, ~, and K (Yu-Lee and Rosen 1983; Jones et al. 1985; Thompson et al. 1985). The casein genes all map to a single chromosome in rodents, sheep, cows, humans, and pigs (reviewed by Mercier and Villotte, 1993) and are very tightly linked genetically. All four bovine casein genes have been mapped to a single 250-kb locus (Ferretti et al. 1990; Threadgill and Womack 1990). This close linkage might be expected in the case of the evolutionarily closely related calcium-sensitive caseins, but there is no evidence that K casein is evolutionarily related to the other caseins. Both in sequence homology and protein function it appears to be related to -/ fibrinogen (Jolles et al. 1974; Thompson et al. 1985; Alexander et al. 1988), which performs a cleavage-induced clotting function in blood similar to the clotting function of K casein in the stomach. Therefore, the proximity of the bovine K casein gene to the other casein genes is noteworthy. Two yeast artificial chromosome (YAC) clones bearing the five murine caseins were obtained by screening the Imperial Cancer Research Fund mouse genomic YAC library, primarily with two genomic probes for murine [3 casein as described (Cox et al. 1993) and secondarily by screening [3 casein-positive clones with cDNA probes for murine e~, 7, e, and K casein, by colony hybridization and pulsed field gel electrophoresis (PFGE). The two c lones are I C R F y 9 0 2 G 0 7 8 1 , r enam ed MP12, and ICRFy902CI 1116, renamed MPI4. MP14 is approximately 380 kb in size, and MP12 is approximately 435 kb in size, as determined by PFGE and Southern blotting (data not shown). A restriction map of the two YACs was generated by partial digestion with PmeI, SaII, XhoI and ClaI, and probing a single filter with probes specific to the left and right arms of pYAC4 (LA and RA; Fig. 1). The restriction fragments detected by LA and RA are listed in Table 1. When the MP12 and MP14 PmeI band patterns are represented as maps, the two maps can be aligned so that the 121-kb MP14 band is lined up with the 105-kb MP12 band. The alignment is almost perfect over the region of overlap, varying by less than 5 kb. By this analysis, the 26-kb MP14 band should fall some 10 kb from the left telomere of MP12 and therefore give a band in the MP12 lanes, but, given that there are 6 kb

Transgenes Display Stable Patterns of Expression in Apple Fruit and Mendelian Segregation in the Progeny

We report here the stable expression and Mendelian segregation of transgenes in a tree species. We present physiological, biochemical, and molecular evidence for stable transgene expression of both nopaline synthase (nos) and the cotransferred gene neomycin phosphotransferase (nptII) in the flesh of apple fruit 7 years after the initial transformation. Evidence based on callus bioassays, gene expression assays, Southern blotting, and PCR data show a 1:1 segregation among 74 seedlings of the R1 progeny for the nos gene but a 3:1 segregation for the cotransferred nptII gene among a subpopulation of 47 seedlings. Southern blot data and the existence of a significant population of seedlings that possessed the nptII gene but not the nos gene have led us to propose the existence of two nptII loci and one nos locus in the original transgenic tree.

The nodule-specific VfENOD-GRP3 gene encoding a glycine-rich early nodulin is located on chromosome I of Vicia faba L. and is predominantly expressed in the interzone II-III of root nodules

A nodule-specific cDNA was isolated from a Vicia faba L. nodule cDNA library. Since time course experiments revealed an early expression of this transcript in the nodule, this cDNA coded for an early nodulin and was designated VfENOD-GRP3. Based on tissue print hybridizations, we found a predominant expression of VfENOD-GRP3 transcripts in the interzone II-III region of broad bean root nodules. The encoded early nodulin ENOD-GRP3 was characterized by an N-terminal signal peptide and a C-terminal domain displaying a glycine content of 31%. Sequence analysis of a genomic VfENOD-GRP3 clone revealed that the signal peptide and the glycine-rich domain were specified by two separate exons. Primer extension experiments identified two adjacent transcription start sites for VfENOD-GRP3 transcripts. The common nodulin sequences 'AAAGAT' and 'CTCTT' were present five and three times on both DNA strands of the putative VfENOD-GRP3 promoter, respectively. Additionally, three sequence motifs resembling organ-specific elements of the soybean lbc3 gene promoter and a sequence similar to the binding site 1 for the nodule trans-acting factor Nat2 were identified. From Southern blot data and from sequence analysis of genomic PCR fragments, the presence of a VfENOD-GRP3 gene family was inferred. By PCR experiments using sequence-specific primers and DNA of microisolated chromosomes as a template, this family was located on the long arm of chromosome I.

Analysis of the gene for the elongation factor lα from the zygomycete Absidia glauca.Use of the promoter region for constructions of transformation vectors

The complete genomic DNA sequence was determined for one of the gene for the elongation factor lα (TEF), isolated from the zygomycete Absidia glauca. Sequence comparison with TEF genes from other fungi show the highest similarity to TEF-genes of the closely related zygomycete Mucor racemosus (Sundstrom et al. 1987). Southern-blot analysis of genomic DNA from A. glauca with the TEF gene reveals six chromosomal copies in the genome. In transformation experiments of A. glauca, vector constructions were used which allow targeting of one of the TEF loci. Several transformants of A. glauca were analyzed at the DNA level. In most cases, rearranged forms of autonomously replicated plasmids could be found in these isolates. However, some transformants show a different restriction pattern of the TEF loci if compared with the parental strains. From Southern-blot data it could be concluded that in one case the rearrangement lies downstream of one TEF locus. In a second case genetic parts following the 3′-end of the TEF gene are moved towards the 5′-end of the gene.

Characterization of a Cα gene of swine

The cDNA sequence encoding the constant region of the porcine IgA heavy chain as well as the exon-intron structure of the germline gene, have been determined. A cDNA clone (1A1) spanning the CH3 domain and part of the CH2 domain was isolated from a porcine mesenteric lymph node cDNA library. Clone 1A1 was aligned with a PCR-generated DNA fragment encompassing the CH1 domain through the 5' end of the CH3 domain to derive the complete cDNA sequence. Comparison with other mammalian Cα heavy chains (hinge regions excluded) indicated that the deduced amino acid sequence of porcine Cα is most homologous with the human Cα subclasses (> 70%), followed by mouse Cα (61%) and a consensus sequence of the 13 rabbit Cα heavy chains (59%). The greatest sequence homology was found among the CH3 domains in all species. A striking feature of porcine Cα is its short six amino acid hinge which like other mammalian IgAs, is encoded with the CH2 domain. Sequence analysis of germline Cα, generated by PCR from liver or sperm DNA, revealed an exon-intron organization similar to other mammalian Cα genes. Genomic Southern blot data are consistent with the presence of a single Cα gene within the porcine genome. Data obtained in these studies will be valuable in pursuing the use of swine as a model in immunological research.

The cryptomonad histone H4-encoding gene: structure and chromosomal localization.

Cryptomonads are unicellular flagellates whose plastids are surrounded by four membranes. A periplastidal compartment, containing eukaryote-type ribosomes, starch grains and a so-called nucleomorph, is located between the inner and outer membrane pairs. The nucleomorph has been shown to be the vestigial nucleus of a eukaryotic endosymbiont. In order to obtain more information about the chromatin structure of the nucleomorph and the host nuclear chromosomes, we studied the distribution of the histone, H4. H4 was not detectable in the nucleomorph by immunolocalization, thus supporting earlier findings by Gibbs [In: Wiesner et al. (Eds.), Experimental Phycology 1, 1990, pp. 145-157]. Likewise, no H4 DNA was demonstrable in the nucleomorph by Southern hybridization. Sequence analysis, and Southern and Northern blot data of a cryptomonad gene, H4, indicate an intermediate position for these genes between animals and plants.

Sequence and genomic organization of the hsp70 genes of Leishmania amazonensis

The sequence and genomic organization of hsp70 genes in Leishmania amazonensis were examined. Maps of overlapping cosmid clones revealed that seven L. amazonensis hsp70 genes are organized into a 24-kb locus containing 3.5-kb tandem repeats. Cosmids covering a different chromosomal region indicated that an eighth hsp70 sequence is located at a distant site. Southern blot data suggested the existence of additional hsp70 genes or pseudogenes. One complete 3.5-kb genomic repeat unit, including coding and intergenic regions, was sequenced. The predicted L. amazonensis HSP70 protein had approximately 95% sequence identity with Leishmania donovani or Leishmania major HSP70, 81–85% identity with trypanosome HSP70, and 68 or 72% identity with human HSP70 or HSP70 cognate, respectively. The GGMP tetrapeptide repeat found in other trypanosomatid HSP70 proteins is absent from the L. amazonensis sequence. Intergenic sequences of L. amazonensis and L. major differed mainly in the presence of short gaps in the L. amazonensis sequence. Potential regulatory heat shock elements were identified in the upstream sequence. Several cDNA clones were also isolated, and two different poly(A) addition sites 100 nucleotides apart were identified.

Sequence, expression pattern, intracellular localization, and targeted disruption of the Dictyostelium myosin ID heavy chain isoform

The complete sequence of the Dictyostelium myosin ID (DMID) heavy chain isoform has been determined from cDNA and genomic clones. Like the DMIB isoform characterized previously, the DMID isoform is up-regulated during starvation-induced chemotactic aggregation, and its 124-kDa heavy chain contains the tail domain sequences that correspond to both the membrane and second actin-binding sites. An antibody that is specific for the DMID isoform was found to stain the actin-rich pseudopods at the leading edge of migrating cells. Protein microsequencing data reveals that the myosin I isoform localized to leading edge pseudopods in a previous study (Fukui, Y., Lynch, T. J., Brzeska, H., and Korn, E. D. (1989) Nature 341, 328-331) was DMIB, indicating that DMID and DMIB also colocalize and that both should influence the dynamics of actin-rich cortical structures. This and other data indicate that the DMID and DMIB isoforms are closely related and are distinct from the DMIA and DMIE isoforms, which possess truncated tail domains and are not up-regulated during chemotactic aggregation. Cells in which the DMID gene was rendered nonfunctional by targeted gene disruption do not show obvious behavioral defects, suggesting that another myosin I isoform(s) (possibly DMIB) might compensate for DMID. Finally, Southern blot data indicate that Dictyostelium may contain as many as nine myosin I isoforms.

A Large Duplication in the Gene for Lysyl Hydroxylase Accounts for the Type VI Variant of Ehlers-Danlos Syndrome in Two Siblings

Ehlers-Danlos syndrome is a heterogeneous disorder characterized by joint hypermobility, skin hyperextensibility, fragility, and other signs of connective tissue involvement. In addition to these, the type VI variant of the disease has some special characteristics such as kyphoscoliosis and ocular abnormalities. The biochemical abnormality in most patients with this autosomal recessively inherited type VI variant is a deficiency in the activity of lysyl hydroxylase (EC 1.14.11.4), the enzyme catalyzing the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. The type VI variant of Ehlers-Danlos syndrome was first identified in two sisters with a reduced amount of lysyl hydroxylase activity in their skin fibroblasts (S. R. Pinnell, S. M. Krane, J. E. Kenzora, and M. J. Glimcher (1972) N. Engl. J. Med. 286: 1013-1020). Our recent molecular cloning of lysyl hydroxylase has now made it possible to study the mutations leading to the deficiency in lysyl hydroxylase activity in these cells. Our data indicate that the mRNA for lysyl hydroxylase produced in the affected cells is about 4 kb in size, whereas it is 3.2 kb in the control cells. The sequencing of the cDNA for lysyl hydroxylase from the affected cells revealed an apparently homozygous duplication rearrangement of nucleotides 1176 to 1955, corresponding to amino acids 326 to 585 in the normal sequence. From Southern blotting data, the duplicated area in the gene equals about 6-9 kb and corresponds to seven exons.

Analysis of the duplicated human C4/P450c21/X gene cluster

Gene duplications, deletions and rearrangements occur with an unusually high frequency in the region of the P450c21 genes encoding 21-hydroxylase. In the human genome, the locus contains at least 6 genes, oriented 5′ C4A, P450c21A, XA, C4B, P450c21B, XB 3′. Sequence analysis of the XA gene, of the 5′ flanking DNA of the C4A gene, and of part of the XB gene revealed that this gene cluster was duplicated by nonhomologous recombination at a CAAG tetranucleotide. The location of this duplication suggests that it may have occurred after mammalian speciation. The XA gene is abundantly expressed in the human adrenal as a stable 2.6 kb RNA, but it is not known if that RNA serves a biological function. Knowledge of the anatomy of the XA gene facilitates genetic analysis of disease-causing lesions in the P450c21B gene. Southern blotting data show that about 76% of disordered P450c21B alleles bear gene microconversions that resemble point mutations; the remaining alleles are equally distributed between gene deletions and large gene conversions.

Distinct genetic groups of Giardia intestinalis distinguished by restriction fragment length polymorphisms.

The taxonomic status of the parasitic protozoal species Giardia intestinalis depends on the morphological similarity of all Giardia isolated from humans and the presumption that Giardia are host-specific. On the basis of electrophoretic data derived from examination of 26 enzyme loci in Australian isolates, it has been proposed that G. intestinalis is a species complex comprising three or four genetically distinct (but morphologically cryptic) species. These received the tentative designations of genetic groups I-IV (R. H. Andrews, M. Adams, P. F. L. Boreham, G. Mayrhofer & B. P. Meloni. International Journal for Parasitology 19, 183-190, 1989). In the present study, two unrelated DNA probes (one specific for a gene encoding a trophozoite surface protein, the other detecting a non-coding repetitive sequence within the G. intestinalis genome) were used in Southern hybridization analyses to examine 10 axenic isolates of G. intestinalis, established from diverse geographical regions in Australia, together with the Portland-1 isolate from the USA. Both probes identified every isolate unambiguously as belonging to one or other of two genetic clusters. Electrophoretic analysis of the same samples indicated that these clusters correspond to the previously defined genetic groups I and II. No heterogeneity was apparent within the seven group I isolates using either probe. However, when probed with the repetitive sequence, the four isolates belonging to group II exhibited small differences in banding patterns, suggesting that this group may be less homogeneous than group I.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphism and deficiency of human factor H-related proteins p39 and p37.

We described previously cDNA clones representing a novel factor H-related 1.4 kilobase mRNA. This mRNA species codes for a doublet of serum proteins of M(r) 39,000 and 37,000 (p39/p37). The respective recombinant proteins of the three clones H-69, pFH1.4a, and pFH1.4b differ in the expression of the epitope recognized by the monoclonal antibody (mAb) 3D11. This probably reflects the difference of three amino acid residues of the deduced protein sequence. Here we report evidence for corresponding alterations in the native proteins p39/p37 in human sera. Employing mAb 3D11 and a polyclonal factor H-specific antiserum we detected three different patterns in western blot analyses of human sera which we provisionally termed FH1.4p+m+, FH1.4p+m-, and FH1.4p-m-. In the first pattern, p39/p37 were recognized by both antibodies, while in the second pattern the two proteins reacted only with the polyclonal antiserum. Both antibodies failed to detect p39/p37 in the third pattern. These phenotypes are found in the healthy population with frequencies of 0.556, 0.40, and 0.044, respectively. The frequencies of the alleles FH1.4*p+m+, FH1.4*p+m-, and FH1.4*p-m- were estimated to be 0.33, 0.46, and 0.21, respectively, assuming the gene distribution to be in Hardy-Weinberg equilibrium. Studies of 98 members from 27 families revealed an autosomal Mendelian inheritance. Southern blot data support our assumption of a polymorphism of the factor H-related proteins p39 and p37.

Characterization of rps17, rp19 and rpl15: three nucleus-encoded plastid ribosomal protein genes.

Approximately two-thirds of the 55 to 60 plastid ribosomal proteins are encoded in the nucleus. Since the protein products of each of these genes are needed in equal amounts for ribosome assembly, their expression may be coordinately regulated by common mechanisms. To begin to understand how the expression of these genes is regulated, we have isolated cDNA and genomic clones for three plastid ribosomal protein genes from an Arabidopsis thaliana library. The genes rps17, rpl9 and rpl15, encoding plastid ribosomal proteins CS17, CL9 and CL15, respectively, are located in the nuclear genome and Southern blot data suggest that each is a single copy gene in A. thaliana. Northern blot data show that transcripts from rps17, rpl9 and rpl15 are much more abundant in leaves and stems than they are in roots. The nucleotide sequences of each of these three genes were determined and their transcriptional initiation sites identified. rps17 transcripts have multiple 5' ends suggesting that they are initiated at multiple sites or are post-transcriptionally processed at their 5' end. rpl9 and rpl15 apparently have unique transcriptional initiation sites but are post-transcriptionally processed to remove six and three introns, respectively, from their primary transcripts. We have examined the genomic sequences for motifs that may be important for the proper expression of these genes. A 7 bp sequence motif, whose consensus is 5'-AGGCCCA-3', flanked by AT-rich regions was identified between 38 and 73 nucleotides upstream of the rps17, rpl9 and rpl15 transcriptional initiation sites.